Metabolomics in Models of Cardiovascular Disease UAB Metab cla… · Metabolomics in Models of Cardiovascular Disease Wednesday, March 15, 2017 Adam R. Wende, Ph.D. Assistant Professor

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GBSC 724: Advanced Topics in Metabolomics

Metabolomics in Models of Cardiovascular Disease

Wednesday, March 15, 2017

Adam R. Wende, Ph.D.

Assistant ProfessorDivision of Molecular and Cellular Pathology

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Outline

• Define the question and model to determine the connection between metabolism and diabetic heart disease.

• Identify the molecular mechanisms by which glucose directly alters molecular function using systems biology.• Transcriptomics• Proteomics• Metabolomics• Epigenetics (e.g. methylomics)

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2.5 million years 50 years

Obesity, Metabolic Syndrome, Diabetes,and Heart Failure

From: Roger Unger - UTSW

ARW www.cdc.gov/diabetes/statistics and www.cdc.gov/mmwr

2010 – Obesity

2010 – Diabetes 2010 – Heart Disease

2010 – Physical Inactivity

≤19% 20%–23% 24%–27% 28%-30% ≥31% 20%-24% 25%–29% ≥30%

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Maintaining Cardiac Function Through Metabolic Substrate Balance

Glucose Fatty Acids

giphy.com

ARW Ungar … Bing 1955 Am J Med 18(3):385

UAB founded in 1969

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Metabolic Substrate Utilization in the Heart

Peterson and Gropler 2010 Circ Cardiovasc Imaging 3:211

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Point/Counterpoint - The Right Balance?

Taegtmeyer and Stanley 2011 J Mol Cell Cardiol 50(1):2

Heinrich Taegtmeyer, MD, DPhil

William C. Stanley, PhD

1957 - 2013

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Diabetes and Metabolomics

Metabolomics is an integral part for understanding diseaseprocesses … information garnered in the biomarkerinvestigations, future research should shed more light ondisease pathogenesis and explore new treatment options.

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Heart failure and substrate switching

The hypertrophy, oxidative stress, and metabolic changesthat occur within the heart when glucose supplants FA as amajor energy source suggest that substrate switching toglucose is not entirely benign.

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Mitochondria – a Dynamic Network

Fan … Brooks 2010 Free Radic Biol Med 49(11):1646

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Mitochondria – too much fat

Serum Serum + 500 μM Palmitate

Adapted from Heiko Bugger

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Fatty Acids

CACAcyl-Carnitine

FAO

Acetyl-CoA

ADP

ATPe-e-

e-

I II III IV

Metabolic Substrate Utilization

H+H+ H+

Glycogensynthase

Hexosamine biosynthetic pathway

UDP-GlcNAc

Acyl-CoA

e-

PDPs

PDKs

GlucoseO

P

GlucoseO

P

Glycolysis

Pyruvate

GLUT1

PDHAcetyl-

CoA

GLUT4

MPC1/2

PFK

HK

GlucoseO

O

TF TF

GLUT4

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Facilitative Glucose Transporters: GLUTs“Solute Carrier Family, SLC2A”

Scheepers … Schurmann 2004 J Parenter Enteral Nutr 28:364

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Changes in Human Heart GLUT Levels

Armoni … Karnieli 2005 J Biol Chem 280(41):34786

Biopsies obtained during coronary bypass surgeryHL = hyperlipidemiaDM2 = diabetes mellitus type 2

Razeghi … Taegtmeyer 2002 Cardiology 280(41):34786

RNAHuman heart failure

ProteinHuman heart diabetes

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Glucose Utilization and Rodent Models of Type 1 Diabetes

Panagia … Clarke 2005 Am J Physiol 288:H2677

ProteinDiabetic Mouse Heart

GLUT4

Glucose UptakeDiabetic Mouse Heart

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Glycolysis GLOX

Belke … Severson 2000 Am J Physiol 279:E1104

Constitutive GLUT4 Expression Prevents Development of Glucose Utilization Defects

Question: Is the change in cardiac metabolic substrate flexibility

adaptive or maladaptive?

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DOX absent = OFF

MHC-rtTArtTAa-MHC

TRE-GLUT4mycGLUT4TRE

DOX present = ON

MHC-rtTArtTAa-MHC


Inducible Cardiomyocyte-Specific GLUT4 Expression (mG4H)

2 4 8

myc

Glut4

DOX (d)

Con

2 4 8

mG4H

0 14 0 14

Con mG4H

14

Hrt

GC

Va

s TA

S

ol

5-fold

2 4 8

myc

Glut4

DOX (d)

Con

2 4 8

mG4H

0 14 0 14

Con mG4H

14

Hrt

GC

Va

s TA

S

ol

5-fold

2 4 8

myc

Glut4

DOX (d)

Con

2 4 8

mG4H

0 14 0 14

Con mG4H

14

Hrt

GC

Va

s TA

S

ol

Heart

Hrt = HeartGC = GastrocnemiusVas = Vastus lateralis

TA = Tibialis anteriorSol = Soleus

mG4H Mice Exhibit Inducible Cardiac-Specific Expression of GLUT4

GLUT4

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Ariel Contreras-FerratWende … Abel in prep

Insulin-induced GLUT4 Vesicle Fusion and Exofacial Myc-Epitope Exposure

Renata O. PereiraWende … Abel in prep

Cardiac Myocytes

2-DG Uptake

GLUT4 Induction Increases Basal and Insulin-Stimulated Glucose Uptake

Basal0.1 nM Ins

n = 3 – 4a P < 0.01 vs. Con-Basalb P < 0.001 vs. All

0

50

100

150

200

250

Con mG4H

pmol

! m

g-1 !

min

-1

b a

a a

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TransgeneInduction

Streptozotocin (STZ)-Induced Hyperglycemia is Not Altered by Transgene Induction

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0

300

600

900

1200

1500

1800

2100

Con mG4H

nmol

! m

in-1

! g

dhw

-1

GLUT4 Induction Increases Glycolysis andRescues Diabetic Cardiac Glycolytic Defects

n = 6 – 10§ P < 0.01 vs. Con

VehicleSTZ

Isolated Working Hearts

Glycolysis

0

300

600

900

1200

1500

1800

2100

Con mG4H

nmol

! m

in-1

! g

dhw

-1

§§

Joseph TuineiWende … Abel in prep

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0

50

100

150

200

250

300

350

Con mG4H

nmol

! m

in-1

! g

dhw

-1

GLUT4 Induction Increases GLOX butAccelerates Diabetic Cardiac GLOX Defects

n = 6 – 10‡ P < 0.001 vs. All* P < 0.01 vs. Veh

VehicleSTZ


Glucose Oxidation (GLOX)

0

50

100

150

200

250

300

350

Con mG4H

nmol

! m

in-1

! g

dhw

-1

*

‡


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GLUT4 Induction Prevents Increased Cardiac POX in Diabetes

VehicleSTZ


Palmitate Oxidation

(POX)

n = 5 – 13‡ P < 0.001 vs. All

0

100

200

300

400

500

600

Con mG4H

µm

ol !

min

-1 !

gdh

w-1

0

100

200

300

400

500

600

Con mG4H

µm

ol !

min

-1 !

gdh

w-1

‡


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Oxidative Phosphorylation

www.genome.jp/kegg/pathway.html

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GLUT4 Induction Accelerates Development of Mitochondrial Dysfunction

n = 3 – 4* P < 0.05

0

20

40

60

80

100

120

Con mG4H

Com

plex

I ac

tivity

(%

of C

on-V

eh)

0

20

40

60

80

100

120

Con mG4H

Com

plex

III a

ctiv

ity

(% o

f Con

-Veh

)

0

20

40

60

80

100

Con mG4H

Com

plex

II a

ctiv

ity

(% o

f Con

-Veh

)

0

20

40

60

80

100

Con mG4H

Com

plex

IV a

ctiv

ity

(% o

f Con

-Veh

)

* ** *

* *

Oleh KhalimonchukWende … Abel in prep

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In the context of diabetes, enhancing glucose delivery by

expression of GLUT4 accelerates the progression of

mitochondrial dysfunction.

Conclusion – Part 1

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Diabetic Cardiomyopathy“Death by a Thousand Cuts…”

ER stress

Adapted from Wende, Symons, and Abel 2012 Curr Hypertens Rep 14(6):517

Inflammation

Mitochondrial dysfunction

REDOX Imbalance

Lipotoxicity

Glucotoxicity

Insulin resistance

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ARW Adapted from Lewis and Abdel-Haleem 2013 Front Physiol 4:237

Systems BiologyTranscriptome Genome

/EpigenomePhenome

Proteome Metabolome

PhenomeObesity, diabetes, heart failure, BHI, etc.

TranscriptomeNortherns, qPCR, microarrayRNA-seq, miR, lncRNA, etc.

ProteomeMass spec, western blot, Co-IP, IHC, PTMs, etc.

MetabolomeGlucometer, ELISA, GC-MS, HPLC, NMR, fluxomics, etc.

Genome / EpigenomeSoutherns, sequencing,GenBank, ENCODE, ChIP-seq, bsDNA-seq, etc.

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Transcriptomic Analysis Using theAgilent SurePrint G3 60K Microarray

181.9 MB

mG4H-Veh

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Pathway Analysis of Microarray

Wende … Abel in prep

-log(p-value)

Metabolic disease

Amino acid metabolism

Lipid metabolism

Nucleic acid metabolism

Carbohydrate metabolism

Skeletal and muscular disorders

Energy production

Cardiovascular system development & function

Post-translational modification

Endocrine system development & function

Inflammatory response

Endocrine system disorders

Cell death

Cardiovascular disease

Gene expression

Nutritional disease

Protein degradation

Threshold0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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Glucose Regulated Gene Expression

Wende, unpublished

0 = up-regulated0 = contra-regulated0 = down-regulated

MouseSTZ

MousemG4H

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Species Conservation of Gene Expression Changes in Diabetes

Drakos … Wende, unpublished

HumanT1D

MouseT1D

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Species Conservation of Gene Expression Changes in Diabetes

Drakos … Wende, unpublished

0.5 1.0 1.5 2.0 0.0 2.5

-log (P-value)

3.0 3.5 4.0

Threshold

Mitochondrial dysfunction

Calcium signaling

3-phosphoinositide degradation

Oxidative phosphorylation

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Oxidative Phosphorylation

GeneSifter using KEGG

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Ndufa9 Gene Promoter Structure

KEYTSS = Transcription start site

= CpG island

= Sp1 RE

http://ecrbrowser.dcode.org

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-2 kb +1 kb

Luc

-0.5 kb

-0.3 kb

TSS

Ndufa9

Ndufa9 Gene Promoter Mapping

C2C12 Myotubesn = 9* P < 0.05

5.5 mM 25 mM

Glucose

0

20

40

60

80

100

-2 kb -0.5 kb -0.3 kb

Nor

mal

ized

RLU

*

* *


Transient Transfection

Promoter Activity

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0

20

40

60

80

100

-2 kb WT -2 kb M3

Nor

mal

ized

RLU



5.5 mM 25 mM

Glucose

*

-2 kb +1 kb

Luc

TSS

Ndufa9 Sp1-M3



Promoter Activity

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0

20

40

60

80

100

-2 kb + (-) -2 kb + Sp1

Nor

mal

ized

RLU



5.5 mM 25 mM

Glucose

*

*

*

*

-2 kb +1 kb

Luc

TSS

Ndufa9

Sp1



Promoter Activity



/EpigenomePhenome

Proteome Metabolome






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O-GlcNAcylation

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Metabolic Integration: Protein O-GlcNAcylation

Hart … Lagerlof 2011 Annu Rev Biochem 80:825

-log(p-value)

Amino acid metabolism

Lipid metabolism

Nucleic acid metabolism

Carbohydrate metabolism

Threshold0 1 2 3 4 5 6 7 8 9 10

Microarray data

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O-GlcNAc Cycling

Hanover … Love 2012 Nat Rev Mol Cell Biol 13(5):312

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GlcNAc Regulation of Sp1

Vosseller … Hart 2002 Curr Opin Chem Biol 6(6):851

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GlcNAcylation Regulates Ndufa9 Gene Expression

0

20

40

60

80

100

120

None GFP OGA

Nor

mal

ized

RLU


5.5 mM 25 mM

Glucose

Li WangWende … Abel in prep


Promoter Activity

0

20

40

60

80

100

120

None GFP OGA

Nor

mal

ized

RLU

*

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Enhanced glucose delivery regulates oxidative capacity

via transcriptional mechanisms including GlcNAcylation of

transcription factors.


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Mitochondrial Protein O-GlcNAcylation and Neonatal Cardiomyocyte Metabolic Function

Hu … Dillmann 2009 J Biol Chem 284(1):547

Mitochondrial Protein O-GlcNAcylation

O-GlcNAcylation of NDUFA9

5.5 mM Glc 30 mM Glc+ Adv (-)

30 mM Glc+ Adv-OGA

Complex I Activity

5.5 mMGlc

30 mM Glc 30 mM Glc+ Adv (-)

30 mM Glc+ Adv-GCA

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2D-PAGE

15%

SD

S-P

AG

E Con-STZ mG4H-Veh

pH 10 pH 3 IEF pH 10 pH 3 IEF

GLUT4 Induction Alters the Cardiac Mitochondrial Glycoproteome

Isolated Mitochondria

2D-PAGEPro-Q

Emerald

Hansjörg Schwertz Wende, unpublished

2D-PAGE

15%

SD

S-P

AG

E Con-STZ mG4H-Veh

pH 10 pH 3 IEF pH 10 pH 3 IEF

Con-Veh Con-STZ

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/EpigenomePhenome

Proteome Metabolome






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Metabolomic Signatures of Diabetic Heart Disease

James CoxGC and HPLC - metabolomics

KEYCon-VehCon-STZmG4H-VehmG4H-STZ

3D – PCA

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ARW Ungar … Bing 1955 Am J Med 18(3):385

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BHB

GLUT4 Induction Alters Cardiac Ketone Utilization Genes

Manoja Brahma

KetoneSynthesis

Acetoacetate

AcAc-CoA

m-Thiolase/Acaa2

Acetyl-CoA

KetoneOxidation

RNA - Microarray-1 1

AcAc-CoA

Acetyl-CoA

HMG-CoA

Acetoacetate

BHB

HMGCS2/Hmgcs2

Fatty acids

CAC

BDH1/Bdh1

SCOT/Oxct1

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ARW Manoja Brahma

GLUT4 Induction Alters Cardiac Ketone Protein GlcNAcylation

GlcNAc

SCOT1

Overlay

Input IP– SCOT1

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Enhanced cardiac glucose delivery alters metabolic flux through other pathways and regulates the mitochondrial

proteome via O-GlcNAcylation.


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/EpigenomePhenome

Proteome Metabolome






ARW Broad Institute Communications

From Human to Mouse and Back Again

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Role of Epigenetics in Gene Expression

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Epigenetics - Programming DCCT: Diabetes Control and Complications Trial

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Epigenetics - MemoryEDIC: Epidemiology of Diabetes Interventions Trial

Pirola … El-Osta 2010 Nat Rev Endocrinol 6(12):665

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Epigenetics: Transgenerational and Drift

Gut and Verdin 2013 Nature 502:489

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Epigenetic Code

ARW Gräff and Tsai 2013 Nat Rev Neurosci 14(2):97

Chromatin Regulation

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How do metabolites fit in?

Arnaudo … Garcia 2013 Epigenetics Chromatin 6(1):24

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Metabolite Signaling to Chromatin

Gut and Verdin 2013 Nature 502:489

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How does GlcNAc fit in?

Gut and Verdin 2013 Nature 502(7472):489

ARW ucsf.edu

DNA Methylation 101

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Exercise Alters DNA Methylation of Key Metabolic Genes

Low = 40% VO2peak High = 80% VO2peak

Subjects fasted overnight and then consumed a high carbohydrate diet 4 hr prior to exercise.

Barres and Zierath 2012 Cell Metab 15:405

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Diabetes Regulated Cardiac DNA Methylation

Pdk4

= C, = 5mC CpG :

Ndufa9

Con STZ

-552 bp -301 bp

Con STZ *

| |

-523 bp -259 bp | |

Wende, unpublished

Pdk4

= C, = 5mC CpG :

Ndufa9

Con STZ

-552 bp -301 bp

Con STZ *

| |

-523 bp -259 bp | |

Pdk4

= C, = 5mC CpG :

Ndufa9

Con STZ

-552 bp -301 bp

Con STZ *

| |

-523 bp -259 bp | |

Heart, LVn = 10* P < 0.05

TargetedbsDNA-seq

5-mCpG

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Methylation and Expression

GeneSifter and Zymo/UCSC Genome Browser

RNA – microarray

Methylation – genome sequencing Legend:

0% 100%

PDK4

VDAC

Protein – western blotCon mG4H

Veh STZ Veh STZ

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Other Human/Mouse Comparisons

Irvin … Arnett 2014 Circulation 130:565

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Other Human/Mouse ComparisonsMouse

Gene ExpressionConVeh

ConSTZ

mG4HVeh

mG4HSTZ GENE

Mouse DNA Methylation

Wende, unpublished

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Where Does Glycemic Memory Fit In?

DOX absent = OFF

MHC-rtTArtTAa-MHC


DOX present = ON

MHC-rtTArtTAa-MHC


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Metabolomics

Wende, unpublished

Con-STZ mG4H-Veh

mG4H-STZ Con-Veh mG4H-1wk mG4H-2wk

ON OFF

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Glucose Cycling AltersEpigenetic Programming

Legend:

0% 100%

Heart, LV Zymo ResearchWende, unpublished

GenomewidebsDNA-seq

5-mCpG

mG4H-ON

mG4H-OFF

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Background

http://chemistry.uchicago.edu/faculty/faculty/person/member/chuan-he.html

5-hmC Wyatt and Cohen 1952 Nature 170(4338):1072Kriaucioni and Heintz 2009 Science 324(5929):929Tahiliani … Rao 2009 Science 324(5929):930

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How does GlcNAc fit in?

Mariappa … Aalten 2013 EMBO J 32:612

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Cellular glucose fluctuations regulates the epigenome via

histone modifications and controlling the machinery for

DNA methylation.


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Sugar Gumming Up the Works

giphy.com

GLUT4Glucose

OO

GlucoseO

O GLUT4Glucose

OO

GlucoseO

O

GlucoseO

O

GlucoseO

O

GlucoseO

O

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Using combined methylomics, transcriptomics, proteomics, and metabolomics we have

begun to define the mechanism of glucotoxicity.

Overall Summary

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Acknowledgements

Knowledge)that)will)change)your)world)

UAB)

UAB)

Division)of)Molecular)and)Cellular)Pathology)

Thomas J Bailey – UndergradManoja K. Brahma – PostdocMark C. McCrory – Lab ManagerBrenna G. Nye – UndergradMark Pepin – MSTPLamario J Williams – Undergrad

Wende Lab

K99R00 HL111322R00 HL111322-S1

U24 DK076169 AHA 0725064Y JDRF 51002608

Other Colleagues & Mentorspast and present

Oleh Khalimonchuk – UNL

Hansjörg Schwertz

E. Dale AbelJohn C. Schell Joseph Tuineimany others…

UAB CollaboratorsSteve Barnes

John C. ChathamDavid K. CrossmanSteve M. PogwizdMartin E. Young

Stavros G. DrakosNikos A. Diakos

Documents

Metabolomics in Models of Cardiovascular Disease UAB Metab cla… · Metabolomics in Models of Cardiovascular Disease Wednesday, March 15, 2017 Adam R. Wende, Ph.D. Assistant Professor